Polyploid baker&#39;s yeasts having a low temperature inactivation property

ABSTRACT

Polyploid yeasts having an lti property are obtained by sporulating a starting polyploid strain of Saccharomyces cerevisiae, selecting at least one segregant having an lti property, backcrossing the selected segregant at least once with another segregant having an opposite mating type, selecting at least two backcross segregants having an lti property and opposite mating types, crossing the selected backcross segregants at least once, and selecting a polyploid strain having a growth potential, an lti property, and an ability to raise a dough.

This is a continuation of application Ser. No. 07/777029, filed Oct. 16,1991, abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a process for the construction of a strain ofbaker's yeast having an lti property and to a yeast strain constructedby this process.

There are various known processes for constructing strains of baker'syeasts which are based in particular on traditional genetics and whichseek to provide these strains with particular properties useful inbakery.

For example, U.S. Pat. No. 4,547,374 describes the construction byselective hybridizations of a strain of the species Saccharomyces whichis resistant to freezing and which may be used as a baker's yeast in thepreparation of a bread dough intended to be frozen prior to fermentationand baking.

U.S. Pat. No. 4,341,871 describes hybrids of baker's yeast which can bedehydrated, even in pressed form, without excessive loss of theiractivity.

U.S. Pat. No. 4,643,901 describes pure strains of baker's yeast whichare capable of fermenting and raising both sweetened and unsweeteneddoughs and which are obtained by hybridization by protoplasmic fusion of"petite" mutants.

Commercial bakery products intended to be stored in a refrigeratorbefore fermentation and baking are also known. However, these products,such as rolls and croissants for example, contain a chemical raisingagent.

SUMMARY OF THE INVENTION

The problem addressed by the present invention was to provide a processfor constructing strains of baker's yeast and strains thus constructedwhich have an lti property, i.e. a property whereby they are inactivebut survive under refrigeration (lti is the abbreviation for the Englishexpression "low temperature inactive"), and which may be used as abaker's yeast in the production of bakery articles intended to be bakedin an oven just before eating, for example after storage in arefrigerator, in a cold room or on a refrigerated shelf.

To this end, a first embodiment of the process according to theinvention for constructing a strain of baker's yeast having an ltiproperty is characterized in that a haploid strain of saccharomycescerevisiae is subjected to a mutagenic treatment, at least one mutanthaving an lti property is selected and is backcrossed at least once witha wild haploid strain of Saccharomyces cerevisiae having an oppositemating type, at least two backcross segregants having an lti propertyand opposite mating types are selected and are crossed at least once anda diploid strain thus obtained having a growth potential, an ltiproperty and an ability to raise a dough is selected.

In the context of the invention, the expression "growth potential" isunderstood to be a capacity for cultivation in a high yield and withgood productivity by an industrially workable culture process, moreparticularly by the traditional process for culturing baker's yeastknown as the fed batch process (slow and progressive addition of a sugarsolution to a yeast suspension with aeration to avoid the formation ofalcohol during the production of biomass and to maximize the yield).

Similarly, the expression "an ability to raise a dough" is understood tobe a capacity for very slowly transforming a dough at refrigerationtemperature, for example by the very slow production of metabolites,such as CO₂ which can be absorbed by the dough and alcohol which can actas a preservative for the dough, the consequence of this very slowtransformation being that the dough is capable of rising when it isplaced directly in an oven, for example after having been stored in arefrigerator, in a cold room or on a refrigerated shelf.

In a second embodiment of the process according to the invention forconstructing a strain of baking yeast having an lti property, apolyploid strain of Saccharomyces cerevisiae is optionally subjected toa mutagenic treatment and is then sporulated, at least one segreganthaving an lti property is selected and backcrossed at least once withanother segregant of this strain having an opposite mating type, atleast two backcross segregants having an lti property and oppositemating types are selected and crossed at least once and a polyploidstrain thus obtained having a growth potential, an lti property and anability to raise a dough is selected.

It has been possible by this process to construct strains of baker'syeast which have the property of being substantially inactive at theusual refrigeration temperatures, more particularly at temperatures ofthe order of 3° to 9° or 10° C., but of surviving at these temperaturesand subsequently recovering their activity at a higher temperature, forexample of the order of 13° to 14° C.

Accordingly, strains of baker's yeast of this type may be used insteadof a chemical raising agent in the production of bakery articlesintended to be baked in an oven just before eating after refrigeration.They may be used in particular in the production of preformed articles,such as for example rolls, croissants and pizza crusts or a dough to bekneaded in the kitchen, which after refrigeration and baking in an ovenhave organoleptic qualities comparable to those of the same articlesfreshly raised under the effect of a traditional commercial baker'syeast and baked in an oven.

Strains of the type in question may also be used as a temperature abuseindicator in food products to be stored under refrigeration.

DETAILED DESCRIPTION OF THE INVENTION

The process according to the invention may start out either from ahaploid strain of Saccharomyces cerevisiae, such as those formingtraditional laboratory baker's yeasts, or from a polyploid strain ofSaccharomyces cerevisiae, such as those forming traditional commercialbaker's yeasts for example.

Accordingly, in the first above-described embodiment of the processaccording to the invention which starts out from a haploid strain ofSaccharomyces cerevisiae, this strain is subjected to a mutagenictreatment. To this end, cells of this strain may be grown in a YPDmedium containing, for example, 2% glucose, 1% yeast extract and 2%peptone and the cells may be treated with a mutagenic agent such as, forexample, ethyl methanesulfonate (EMS) or ICR-170.

Several mutants having an lti property, more particularly a property ofinactivity but survival at a temperature of the order of 9° or 10° C.,are then preferably selected.

The mutant(s) selected are then backcrossed at least once with a wildhaploid strain of Saccharomyces cerevisiae having an opposite matingtype in order to avoid any unwanted mutations which the starting haploidstrain may have at the outset or after the said mutagenic treatmentand/or to retain if possible an lti property due to only a singlemutation. If several backcross operations are carried out, at least onesegregant having an lti property, more particularly a property ofinactivity but survival at a temperature of approximately 9 or 10° C.,may be selected between two successive operations and the segregant(s)thus selected may be subjected to the second of these two successiveoperations.

At least two backcross segregants having opposite mating types and anlti property, more particularly a property of inactivity but survival ata temperature of approximately 9° or 10° C., are then selected and arecrossed at least once.

Finally, a diploid strain of Saccharomyces cerevisiae thus obtainedhaving a growth potential, an lti property and an ability to raise adough is selected. It is possible in this final stage to retain moresevere and more complete selection criteria than those used in thepreceding stages. More particularly, it is possible to subject thediploid strain(s) to a growth test in the traditional fed batch processfor culturing baker's yeast.

The lti property may then be verified by subjecting the diploidstrain(s) to a CO₂ production test in a maltose-containing nutrientmedium, i.e. in a nutrient medium containing maltose as carbon source,as a function of temperature, for example every day between 3 and 7 d,and as a function of temperature, for example every degree between 3°and 14° C.

Finally, the ability of the strain to raise a dough may be verified byincorporation of the strain as sole raising agent in a pizza dough, forexample by forming pizza crusts with this dough, storing them for a fewdays or even for a few weeks at refrigeration temperature and thenbaking them in an oven. This test may also be completed by verifying theCO₂ production of the strain in maltose-containing medium, for exampleat a temperature of approximately 20° to 30° C.

Accordingly, it is possible by this first embodiment of the processaccording to the invention to construct strains which have a remarkablelti property by virtue of which they may be used in the production ofbakery articles intended to be baked in an oven just before eating afterstorage under refrigeration. More particularly, it is possible toconstruct strains of the baker's yeast Saccharomyces cerevisiae whichhave a growth potential in the fed batch process, an ability to raise adough, a CO₂ production level of less than 20 ml per g pressed yeastafter 7 d in maltose-containing medium refrigerated to 3°-10° C. and aCO₂ production level of at least 40 ml per g pressed yeast after 6 d inmaltose-containing medium kept at a temperature of at least 14° C.

Among the various strains of Saccharomyces cerevisiae thus obtained,three were lodged by way of example at the National Collection ofIndustrial and Marine Bacteria Ltd. (NCIMB), P.O. Box 31, 135 AbbeyRoad, ABERDEEN AB9 8DG, Scotland (United Kingdom) under the BudapestTreaty on the Nov. 6, 1990 and have been given the Nos. NCIMB 40328,40329 and 40330.

In the second embodiment of the process according to the invention,which starts out from a polyploid strain of Saccharomyces cerevisiae,this strain is thus optionally subjected to a mutagenic treatment. Ithas in effect been found that it was not always necessary to subject thestrain to a mutagenic treatment because certain commercial baker'syeasts, for example, can initially show mutations capable of beingdetected in the course of the process according to the invention.

In cases where a polyploid strain is subjected to a mutagenic treatment,it is possible to this end to grow cells of this strain in a YPD mediumcontaining for example, 2% glucose, 1% yeast extract and 2% peptone andthe cells obtained may be treated with a mutagenic agent such as, forexample, ethyl methanesulfonate (EMS) or ICR-70.

Accordingly, in this second embodiment of the process according to theinvention, a polyploid strain of Saccharomyces cerevisiae is sporulatedafter having optionally been subjected to a mutagenic treatment. Tosporulate this strain, cells thereof may be grown for 1 or 2 days on aso-called presporulation medium, such as a PSA medium containing 0.8%yeast extract, 0.3% peptone, 10% glucose and 2% agar. They may then betransferred and kept for 3 to 5 d on a sporulation medium, such as an SAmedium containing 1% potassium acetate, 0.1% yeast extract, 0.05%glucose and 2% agar. The spores may then be isolated, for example bymicromanipulation, and strains of reduced ploidy, in other wordssegregants, may be obtained from them, for example by germination on aYPD medium.

Several segregants of the said polyploid strain having an lti property,more particularly a property of inactivity but survival at a temperatureof approximately 9°-10° C., are then preferably selected. Thesegregant(s) selected is/are then backcrossed at least once with anothersegregant of the polyploid strain which does not have the lti property,but an opposite mating type in order to avoid any unwanted mutationswhich the polyploid strain may have at the outset or after the optionalmutagenic treatment and/or only to retain if possible an lti propertydue to a single mutation. If several backcross operations are carriedout, at least one segregant having an lti property, more particularly aproperty of inactivity but survival at a temperature of approximately 9°to 10° C., may be selected between two successive operations and this orthese segregant(s) may be subjected to the second of these twosuccessive operations.

At least two backcross segregants having opposite mating types and anlti property, more particularly a property of inactivity but survival ata temperature of approximately 9° to 10° C., are then selected and arecrossed at least once.

Finally, a polyploid strain of Saccharomyces cerevisiae thus obtainedhaving a growth potential, an lti property and an ability to raise adough is selected. To this end, the same selection criteria may beretained and the said polyploid strain(s) may be subjected to the sametests as described above in the final stage of the first embodiment ofthe process according to the invention.

Accordingly, it is possible by this second embodiment of the processaccording to the invention to construct strains which have a remarkablelti property by virtue of which they may be used in the production ofbakery articles intended to be baked in an oven just before eating afterstorage under refrigeration. More particularly, it is possible toconstruct strains of the baker's yeast Saccharomyces cerevisiae whichhave a growth potential in the fed batch process, an ability to raise adough, a CO₂ production level of less than 30 ml per g pressed yeastafter 7 d in maltose-containing medium refrigerated to 3°-9° C. and aCO₂ production level of at least 60 ml per g pressed yeast after 6 d inmaltose-containing medium kept at a temperature of at least 13° C.

Among the various strains of Saccharomyces cerevisiae thus obtained, twowere lodged by way of example at the National Collection of Industrialand Marine Bacteria Ltd. (NCIMB), P.O. Box 31, 135 Abbey Road, ABERDEENAB9 8DG, Scotland (United Kingdom) under the Budapest Treaty on the 6thNovember, 1990 and have been given the Nos. NCIMB 40331 and 40332.

EXAMPLES

The process according to the invention and the strains obtained areillustrated by the following Examples, in which percentages and partsare by weight, unless otherwise indicated.

The Examples are preceded by a description of various tests and of thecomposition of the various media used, by a brief description of thevarious Figures of the accompanying drawings and by a ComparisonExample.

TESTS

1. Growth test

To simulate a real growth test in the traditional fed batch process forculturing baker's yeast, a growth test was designed using variousculture media containing non-fermentable carbon sources, such as lacticacid (S-lac medium), ethanol (S-EtOH 2% and S-EtOH 1% media) andglycerol (YPG medium).

The reasoning behind this test is that baker's yeast accumulatesnon-fermentable carbon-containing metabolites in response to a criticaladdition rate of a solution of a fermentable sugar, such as sucrose, inthe course of the fed batch process. These metabolites have aninhibiting and toxic effect on the metabolism and on the respiration ofthe yeast. Strains having a high growth potential can accumulate thesetoxic carbon-containing metabolites at higher addition rates and areless sensitive to the accumulation of these metabolites than strainshaving a lower growth potential.

The results of this growth test are shown in FIG. 1 in terms of thedimensions which the colonies have after incubation of cells of a strainon plates of the said media for a period of 3 d at a temperature of 30°C. The horizontal bands corresponding to each medium stop at marks of 0;0.5 mm; 0.5-1.5 mm; 1.5-2.5 mm; 2.5 mm, indicating a mean observed sizeof the colonies.

2. CO₂ production test

This test is carried out in a specially designed apparatus comprising atemperature gradient block, for example having cells at varioustemperatures into which the lower end of fermentation tubes may beintroduced. These tubes have a closed and graduated upper end and anexpansion flask connected to one side. The CO₂ produced by the yeastaccumulates in the upper graduated end of each tube, the culture mediumdisplaced by the accumulation of gas being able to pass into theexpansion flask.

To carry out this test, 2 ml of an overnight culture in YPD medium ofthe strain to be tested is inoculated into 200 ml of a first mediumcontaining 0.67% of a nitrogen base without amino acids, such as theproduct marketed by the Difco company under the name "yeast nitrogenbase w/o amino acids" for example, 0.5% yeast extract, 2% sucrose, 1%sodium succinate and concentrated hydrochloric acid to adjust the pH to4.5 in a 500 ml flask. The whole is incubated with stirring for 24 h at30° C.

The cells are separated by centrifugation at 6,000 g/20° C. for 5minutes and are then suspended in 200 ml of a second medium containing0.67% of a nitrogen base without amino acids, 0.3% yeast extract and0.3% sucrose, 1% sodium succinate and concentrated hydrochloric acid toadjust the pH to 4.5 in a 500 ml flask. The whole is incubated for 24 hat 30° C.

The cells are separated by centrifugation at 6,000 g/4° C. for 5 minutesand the residue of yeast cells obtained is washed twice with 50 mldistilled water.

The cells are suspended in 10 ml distilled water and are transferred to15 ml graduated and preweighed polypropylene tubes. They are centrifugedat 3,000 g/4° C. for 10 minutes. The tubes are drained, the yeastresidues are weighed and are suspended in a quantity of 0.61 g yeastresidue equivalent to 0.5 g pressed yeast having a dry matter content ofapproximately 27% per ml in a third medium containing 0.67% nitrogenbase without amino acids, 2% maltose, 1% sodium succinate andconcentrated hydrochloric acid to adjust the pH to 4.5. Quantities of0.5 ml (for temperatures of ≧10° C.) or 1 ml (for temperatures of <10°C.) are introduced into fermentation tubes of the type described abovewhich have each been filled with 50 ml of the third medium, in otherwords the maltose-containing medium, and cooled to 4° C.

The fermentation tubes are incubated at the desired temperatures in thetemperature gradient block described above. The production of CO₂ isrecorded at selected intervals after the tubes have been immersed for afew seconds in a sonication bath to release CO₂ bubbles retained in theliquid medium.

3. Dough raising test

A dough is prepared by mixing 30 parts water, 60 parts white soft wheatflour, 1.4 parts sodium chloride and 7.6 parts peanut oil. The strain tobe tested is incorporated in the flour in a quantity of 1 part pressedyeast. 20 cm diameter and 0.5 cm thick pizza crusts are then formed fromthe dough and are stored for 21 d at 8° C. in a sealed plastic wrapping.

The pizza crusts are then removed from their wrapping and baked in anoven for 15 minutes at 180° C.

The test is considered to have been passed if the pizza crusts have athickness of approximately 2 cm and organoleptic qualities, namely ataste and texture, comparable with those of analogous pizza Crustsprepared with a dough freshly raised under the effect of a traditionalcommercial baker's yeast.

MEDIA YPD

Glucose 2%

Yeast extract 1%

Peptone 2%

PSA

Glucose 10%

Yeast extract 0.8%

Peptone 0.3%

Agar 2%

SA

Glucose 0.05%

Yeast extract 0.1%

Potassium acetate 1%

Agar 2%

S-lac

0.67% Nitrogen base without amino acids

0.5% Lactic acid

2% Agar

S-EtOH 1% or 2%

0.67% Nitrogen base without amino acids

1% or 2% Ethanol

2% Agar

YPG

Glycerol 2%

Yeast extract 1%

Peptone 2%

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a linear representation of the size of the colonies obtainedby growth on S-lac, S-EtOH 1% and 2% and YPG media of cells of thestrains NCIMB 40328, 40329, 40330, 40331, 40332 and "levure boulangjerebleue" (LBB, for comparison). Each of FIGS. 2 through 7 is,respectively, a three-dimensional representation of the CO₂ productionlevel in maltose medium as a function of the temperature and time forthe strains NCIMB 40328 (FIG. 2), NCIMB 40329 (FIG. 3), NCIMB 40330(FIG. 4), NCIMB 40331 (FIG. 5), NCIMB 40332 (FIG. 6) and "levureboulangere bleue" (LBB for comparison, FIG. 7).

FIG. 8 is a two dimensional representation of the CO₂ production levelin maltose medium at 30° C. as a function of time for the strains NCIMB40328, 40329, 40330 and 40332 and "levure boulangere bleue" (LBB forcomparison).

Comparison Example

By way of comparison, a strain of commercial baker's yeast, namely astrain forming the baker's yeast marketed by the Fould Springer companyunder the name "levure boulangere bleue" (LBB), is subjected to the sametests as those used above to select the said diploid and polyploidstrains having a growth potential, an lti property and an ability toraise a dough, i.e. to tests 1 to 3 described above.

The results of the comparative growth test presented in FIG. 1 show asexpected that the LBB strain has a very good growth potential in thetraditional fed batch process for culturing baker's yeast.

The results of the comparative CO₂ production test are presented in FIG.7 where it can be seen that the production of CO₂ in maltose mediumexceeds 40 ml per g pressed yeast after 6-7 d at 3°-5° C., after 5-6 dat 5°-9° C. and after 3-4d at10° C.

It can also be seen from FIG. 8 that the CO₂ production in maltosemedium of the LBB strain rapidly exceeds 20 ml per g pressed yeast afterapprox. 4 h at 30° C.

The results of the comparison test for raising a dough show that the LBBstrain does not lend itself to refrigerated storage before baking in anoven. The sealed plastic wrapping in which a pizza crust obtained usingthe LBB strain is wrapped inflates like a balloon after 1 d at 8° C.

By contrast, it can be seen that there is hardly any discernibledifference in quality between the pizza crusts obtained using strainsconstructed by the process according to the invention and subjected tothe above dough raising test on the one hand and the pizza crustobtained using the LBB strain and subjected to the same test without therefrigerated storage phase.

Example 1

The starting strain is a haploid strain of Saccharomyces cerevisiae,such as those forming traditional laboratory baker's yeasts, moreparticularly a strain having the genotype MATa arg4-17 his4-38 lys1--1met8-1 trpl-1 mal, which was lodged under the Budapest Treaty on the06.11.90 at the National Collection of Industrial and Marine BacteriaLtd. (NCIMB), P.O. Box 31, 135 Abbey Road, ABERDEEN AB9 8DG, Scotland(United Kingdom), where it has been given the No. NCIMB 40333.

This strain is subjected to a mutagenic treatment with EMS. To this end,cells of this strain are grown in 5 ml of YPD culture medium up to thestationary phase, washed once with a 100 mM potassium phosphate bufferat pH 7.0 and then suspended in the same buffer in a quantity of 10⁸cells/ml.

3% by volume EMS is added to the suspension which is then left to reactfor 1 h at 30° C. with vigorous stirring. The treatment is terminated bydilution of the suspension in 10 times its volume of a 5% solution ofsodium thiosulfate. The cells are then distributed over solid YPD mediumand are cultured thereon for 2 d at 30° C. They are then redistributedover solid YPD medium and cultured thereon for 3 weeks at 10° C. to testtheir lti property.

Some stable mutants having the property of inactivity but survival atthat temperature are then selected.

One of these mutants having a mating type MATa is then backcrossed witha wild haploid strain of Saccharomyces cerevisiae such as, for example,the strain GRF18 having the genotype MATα canl his3-11,15 leu2-3,112,which is well known to the expert (cf. G. R., Fink, Whitehead Institute,Nine Cambridge Center, Cambridge, Mass. 01 142, USA).

A segregant of this backcross having a pronounced lti property at 10° C.and the genotype MATa lys1 his 3/4 trp1 mal is then selected.

This segregant is backcrossed with the same wild strain as that used forthe backcross of a mutant of the starting strain.

Two segregants of this backcross showing a pronounced lti property at10° C. and each having the genotype MATα leu2 his3/4 mal are thenselected.

Each of these two segregants is backcrossed with a wild haploid strainof Saccharomyces cerevisiae having at least one MAL gene such as, forexample, the strain 1403-7A which is well known to the expert (cf. YeastGenetics Stock Center, 6th Edition of the Catalog by Robert Mortimer,Department of Biophysics and Medical Physics, University of California,Berkeley, Calif. 94720, USA).

A segregant of each of these backcrosses each with a very pronounced ltiproperty at 10° C. is then selected, one having a genotype MATa MAL andthe other a genotype MATα leu2 MAL.

These two segregants are crossed and various diploid strains ofSaccharomyces cerevisiae produced by this crossing, which have a growthpotential, an lti property and an ability to raise a dough, areselected.

Among the various strains thus obtained, the above-mentioned strainNCIMB 40328 has been lodged by way of example. This strain has arelatively modest growth potential in the traditional fed batch processfor culturing baker's yeast, as can be seen from FIG. 1 whichillustrates the results obtained in the above growth test.

However, it shows a high capacity for fermenting a dough because iteasily passes the corresponding test described above.

Finally, it has a prounced lti property, as can be seen from FIG. 2which is a three dimensional diagram showing its CO₂ production level asa function of the fermentation temperature and time in maltose medium,as determined by the CO₂ production test described in detail in theforegoing. It can be seen that it is substantially inactive between 3°and 10° C. for at least about 1 week, but survives and is capable ofreturning to significant activity after about 6 to 7 d at approximately13° to 14° C. More particularly, it can be seen that its CO₂ productionlevel is still around zero after 7 d between 3 and 8° C. and that itonly rises to approximately 8 ml per g pressed yeast after 7 d at 10° C.By contrast, its CO₂ production level in maltose medium increases tomore than 40 ml/g after 6 d at 13° to 14° C.

It can also be seen from FIG. 8 that its CO₂ production level in maltosemedium increases rapidly to more than 20 ml/g after approximately 2 h at30° C.

In addition, the strain NCIMB 40328 shows the following characteristics:

Morphology

Elliptical cells. Certain cells increase in size and form apseudomycelium.

Fermentation of sugars

The strain is capable of fermenting sucrose, maltose and glucose.

Example 2

The starting strain used and the procedure are both as described inExample 1 up to selection of the two backcross segregants having apronounced lti property at 10° C. and the genotype MATα leu2 his3/4 mal.

One of these two segregants is backcrossed with a wild haploid strain ofSaccharomyces cerevisiae such as, for example, the strain X2180-1Ahaving the genotype MATa CUP1 SUC2 ga12 mal mel which is well known tothe expert (cf. Yeast Genetics Stock Center, 6th Edition of theCatalog).

A segregant of this backcross having a pronounced lti property at 10° C.and the genotype MATα his3/4 leu2 ma1 is selected.

This segregant is backcrossed with the same strain X2180-1A as above toeliminate the auxotrophic mutations his3/4 and leu2.

A segregant of this backcross having the genotype MATα mal and asignificant lti property at 10° C. due to a single mutation, in otherwords having a perfect 2:2 segregation in regard to the lti property, isselected.

This segregant is backcrossed with a wild haploid strain ofSaccharomyces cerevisiae having at least one MAL gene such as, forexample, the strain 1403-7A which is well known to the expert (cf. YeastGenetics Stock Center, 6th Edition of the Catalog).

On the one hand, one segregant of this backcross having a pronounced ltiproperty at 10° C. and the genotype MATα MAL and, on the other hand, twosegregants of this backcross respectively showing pronounced and verypronounced lti properties at 10° C., each with the phenotype MATa ura3mal, are selected.

Each of these two last segregants is crossed with the first and variousdiploid strains of Saccharomyces cerevisiae produced by these twocrossings, which have a growth potential, an lti property and an abilityto raise a dough, are selected.

Among the various strains thus obtained, the above-mentioned strainsNCIMB 40329 and NCIMB 40330 each produced by one of these two crossingshaving been lodged by way of example. These two strains have arelatively good growth potential in the traditional fed batch processfor culturing baker's yeast, as can be seen from FIG. 1.

They also have a good ability to raise a dough because they easily passthe corresponding test described above.

Finally, they have a marked lti property. Thus, it can be seen fromFIGS. 3 and 4 that they are substantially inactive in maltose mediumbetween 3° and 10° C. for at least about 1 week, but survive and arecapable of returning to significant activity after about 5 to 7 d atapproximately 11° to 14° C. More particularly, it can be seen that theirCO₂ production level is still around zero after 7 d between 3° and 9° C.and that it only rises to approximately 12 ml per g pressed yeast after7 d at 10° C. By contrast, their CO₂ production level increases to morethan 40 ml/g after 6 d at 12° to 14° C.

It can also be seen from FIG. 8 that their CO₂ production level inmaltose medium increases rapidly to more than 20 ml/g afterapproximately 1.5 h at 30° C.

In addition, these strains show the following characteristics:

NCIMB 40329 Morphology

Elliptical cells. Cells of relatively homogeneous size.

Fermentation of sugars

Capable of fermenting sucrose, maltose and glucose.

NCIMB 40330 Morphology

Elliptical cells. Cells of relatively homogeneous size.

Fermentation of sugars

Capable of fermenting sucrose, maltose and glucose.

Example 3

The starting strain is a commercial polyploid strain of Saccharomycescerevisiae such as that forming the baker's yeast marketed by the FouldSpringer company under the name "levure boulangere bleue", of which thecells contain three to four times more DNA than the cells of a haploidstrain of Saccharomyces cerevisiae.

To sporulate this strain, its cells are grown on a PSA presporulationmedium for 2 d at 30° C. They are then transferred to an SA sporulationmedium where they are kept for 4 d at 30° C.

Spores are then isolated and germinated on a YPD culture medium toobtain strains, i.e. segregants, having reduced ploidy.

One of these segregants having a very pronounced lti property at 10° C.and, in addition, the mating type MATα is selected.

This segregant is backcrossed with another of the segregants of reducedploidy which does not show an lti property, but does have the matingtype MATa.

Several segregants of this backcross which have a very pronounced ltiproperty at 10° C. and one mating type are selected.

Segregants having opposite mating types are crossed several times andvarious polyploid strains of Saccharomyces cerevisiae produced by thesecrossings, which have a growth potential, an lti property and an abilityto raise a dough, are selected.

Among the various strains thus obtained, the above-mentioned strainsNCIMB 40331 and NCIMB 40332 have been lodged by way of Example. Moreparticularly, these two strains have a very good growth potential in thetraditional fed batch process for culturing baker's yeast, as can beseen from FIG. 1.

They also have a good ability to raise a dough because they easily passthe corresponding test described above.

Finally, they have a pronounced lti property. Thus, it can be seen fromFIGS. 5 and 6 that they are substantially inactive in maltose mediumbetween 3° and 9° C. for at least 4 to 5 d, but survive and are capableof returning to significant activity after about 5 to 7 d atapproximately 10° to 13° C. More particularly, it can be seen that theirCO₂ production level is still around zero after 5 d between 3° and 9° C.and that it only rises to approximately 20 to 25 ml per g pressed yeastafter 7 d at 3° to 9° C. By contrast, their CO₂ production levelincreases to more than 60 ml/g after 5 d at 12° to 14° C.

It can also be seen from FIG. 8 that the CO₂ production level of thestrain NCIMB 40332 in maltose medium increases rapidly to more than 20ml/g after approximately 4 h at 30° C.

In addition, these strains have the following characteristics:

NCIMB 40331 Morphology

Round cells. Cells of homogeneous size.

Fermentation of sugars

Capable of fermenting sucrose, maltose and glucose.

NCIMB 40332 Morphology

Round cells. Cells of homogeneous size.

Fermentation of sugars

Capable of fermenting sucrose, maltose and glucose.

We claim:
 1. A process for constructing a strain of baker's yeastcomprising:sporulating a starting polyploid strain of Saccharomycescerevisiae to obtain segregants having reduced ploidy; selecting atleast one segregant having a CO₂ production level of less than 30 ml perg pressed yeast after seven days in maltose medium refrigerated at from3° C. to 9° C. and a CO₂ production level of at least 60 ml per gpressed yeast after 6 days in maltose medium maintained at a temperatureof at least 13° C.; backcrossing a selected segregant at least once withanother segregant having an opposite mating type; selecting at least twobackcross segregants having opposite mating types and a CO₂ productionlevel of less than 30 ml per g pressed yeast after seven days in maltosemedium refrigerated at from 3° C. to 9° C. and a CO₂ production level ofat least 60 ml per g pressed yeast after 6 days in maltose mediummaintained at a temperature of at least 13° C.; crossing the selectedbackcross segregants at least once to obtain polyploid strains; andselecting a polyploid strain having growth potential, ability to raise adough, and a CO₂ production level of less than 30 ml per g pressed yeastafter seven days in maltose medium refrigerated at from 3° C. to 9° C.and a CO₂ production level of at least 60 ml per g pressed yeast after 6days in maltose medium maintained at a temperature of at least 13° C. 2.The process according to claim 1 further comprising subjecting thestarting polyploid strain of Saccharomyces cerevisiae to a mutagenictreatment before sporulating.
 3. The process according to claim 2wherein the starting polyploid strain is treated with ethylmethanesulfonate or ICR-70.
 4. The process according to claim 1 whereinthe starting polyploid strain is sporulated by growing cells on a PSApresporulation medium, transferring the cells to an SA sporulationmedium, and then isolating and germinating spores on a YPD culturemedium.
 5. The process according to claim 1 wherein the selectedsegregant having a CO₂ production level of less than 30 ml per g pressedyeast after seven days in maltose medium refrigerated at from 3° C. to9° C. and a CO₂ production level of at least 60 ml per g pressed yeastafter 6 days in maltose medium maintained at a temperature of at least13° C. is backcrossed with a segregant which does not have a CO₂production level of less than 30 ml per g pressed yeast after seven daysin maltose medium refrigerated at from 3° C. to 9° C. and a CO₂production level of at least 60 ml per g pressed yeast after 6 days inmaltose medium maintained at a temperature of at least 13° C.
 6. Theprocess according to claim 1 wherein the selected backcross segregantsare crossed several times.